The statements in the Background are not necessarily meant to endorse the characterization in the cited references.
Telomeres, the tips of eukaryotic chromosomes, protect the chromosomes from nucleolytic degradation, end-to-end fusion, and recombination. Telomeres are structures at the ends of chromosomes characterized by repeats of the nucleotide sequence (5′-TTAGGG-3′)n. Telomeres shorten as a consequence of normal cell division and critically short telomeres lead to cellular senescence or apoptosis. A rich body of epidemiological and clinical studies in humans in the past decade has linked short telomere length to high risks of aging-related disease and all-cause mortality (Puterman, E. and E. Epel, Soc Personal Psychol Compass, 2012. 6(11) 807-825; Zhu, H., M. Belcher, and P. van der Harst, Clin Sci (Lond), 2011. 120(10) 427-40; and Fyhrquist, F. and O. Saijonmaa. Ann Med, 2012. 44 Suppl 1 S138-42). Genetic, environment, lifestyle, and behavioral factors collectively impact telomere length. Therefore, telomere length has become an index for overall health, disease, and mortality risk.
While average telomere length was measured in almost all the clinical studies published and has demonstrated utility in stratifying patient disease and mortality risk, recent work in mice has also shown that the population of short telomeres is the triggering signal to senescence or apoptosis (Hemann, M. T., et al. Cell, 2001. 107(1) 67-77), and thus disease and mortality risk. In a study reported by Hemann et al, 6th generation telomerase RNA knockout mice (mTR−/−G6) with short telomeres were crossed with mice heterozygous for telomerase (mTR+/−) with long telomeres. The phenotype of the telomerase null offspring mirrors that of the mTR−/− parent despite the fact that half of their telomeres are long, suggesting that the quantity of short telomeres, and not average telomere length, is critical for cell viability and chromosome stability. In people taking a natural product-derived telomerase activator (TA-65®), a significant reduction in the percentage of short (<3 or <4 kbp) telomeres (as measured by a quantitative FISH technology; see (Canela, A., et al. Proc Natl Acad Sci USA, 2007. 104(13) 5300-5) was detected in the leukocytes, although no change in mean telomere length was seen (Harley, C. B., et al., Rejuvenation Res. 2011. 14(1) 45-56). Changes in the percentage of short telomere abundance therefore is expected to be a more sensitive measurement of the effects of lifestyle and pharmacological or other interventions on telomeres. Another study (Vera et al., “The Rate of Increase of Short Telomeres Predicts Longevity in Mammals”, Cell Reports (2012), world wide web URL: dx.doi.org/10.1016/].celrep.2012.08.023) found that “the rate of increase in the abundance of short telomeres was a predictor of lifespan”.
Various methods have been developed for the measurement of telomere length in genomic DNA, including Southern blotting (Kimura, M. et al., Nature Protocols, 2010, 5:1596-1607), Q-FISH (Rufer, N. et al., Nat. Biotechnol., 1998, 16:743-747), flow FISH (Baerlocher, G. M. et al., Cytometry, 2002, 47:89-99), and qPCR (Cawthon, R. M., Nucleic. Acids Res., 2002, 30(10):e47). All of these methods can be used in a clinical setting to monitor health status and permit physicians to prescribe prophylactic or therapeutic intervention tailored to the needs of the individual patient.
To measure the population of short telomeres, quantitative fluorescent in situ hybridization (Q-FISH) of metaphase-spread cells has been used to generate histograms of telomere signal intensities which represent the length of individual telomeres (Poon, S. S., et al., Cytometry, 1999. 36(4) 267-78). Limitations of this method are that live cells are needed, costs are high, and throughput is low. A higher throughput modification of the Q-FISH assay (HTQ-FISH; see Canela, A., et al. Proc Natl Acad Sci USA, 2007. 104(13) 5300-5) was recently championed by the company Life Length (Spain) to measure percentage of short telomeres. Despite the claim, unfortunately, with current technology, this assay cannot be accurate, due to clustering of telomeres, especially short telomeres, in single spots (telomeric associations; see Paeschke, K., K. R. McDonald, and V. A. Zakian. FEBS Lett, 2010. 584(17) 3760-72). Confounding this issue is the fact that short telomeres tend to associate with one another more frequently than long telomeres. In addition, FISH technologies are known to suffer from non-specific binding of the probe to macromolecules in live or fixed cells. A high throughput method to measure percentage of short telomeres that is low-cost and does not require live cells will be much easier to be adapted in both epidemiological and clinical settings, and will have better analytical performance than Q-FISH.
U.S. Pat. No. 5,741,677 (Kozlowski et al.) refers to methods for measuring telomere length. One method involves contacting the telomere with a linker sequence under conditions in which the linker sequence is ligated or otherwise covalently bonded to the 3′ end of the telomere. The telomere sequences are amplified by long PCR amplification with a first primer specific for the linker sequence and a second primer specific for a subtelomeric region of the chromosome. Another method involves preparing DNA extracts of cells, incubating the extract with an oligonucleotide probe complementary to a telomere repeat sequence, and determining amount of probe bound as a measure of telomere length. In addition, a method of measuring telomere length by binding the genomic DNA to a solid phase, and hybridizing the bound DNA with a labeled probe is described.
U.S. Patent Publication No. 2004/0265815 (Baird et al.) refers to a method for measuring telomere length. Baird et al. describes the following steps to detect the length of a population of telomeres: a) annealing the 3′ end of a single-stranded oligonucleotide (hereinafter referred to as a telorette) to a single-stranded overhang of the telomere comprising the G-rich telomere strand (comprising TTAGGG repeat sequences) and covalently binding the telorette to the 5′ end of the C-rich telomeric strand (having CCCTAA repeat sequences), b) amplifying the ligation product formed in step (a) to form a primer extension product; and (c) detecting the length of the primer extension product(s) of step (b). (See also Baird, D. M., et al., Nat Genet., 2003, 33(2):203-7; and Baird D M, Rowson J, Wynford-Thomas D, Kipling D.; Nat Genet., 2003, 33(2):203-7. Epub 2003 Jan. 21. PMID: 12539050)
U.S. Pat. No. 6,514,693 (Lansdorp) refers to a method for detecting multiple copies of a repeat sequence in a nucleic acid molecule in morphologically intact chromosomes, cells, or tissue sections comprising: (a) treating the nucleic acid molecule with a PNA probe which hybridizes to a repeat sequence in the nucleic acid molecule and which is labeled with a detectable substance, under denaturing conditions utilizing a denaturing agent, permitting the probe to hybridize in situ to the repeat sequence in the nucleic acid molecule; and (b) identifying said probe hybridized to the repeat sequence in the nucleic acid molecule by directly or indirectly detecting the detectable substance, thereby detecting the multiple copies of a repeat sequence in a nucleic acid molecule.
Methods of determining short telomere abundance include Southern blot analysis, quantitative fluorescence in situ hybridization (Q-FISH) (Poon, S. S., et al., Cytometry, 1999, 36(4):267-78) and a modified high throughput version of Q-FISH (HT-Q-FISH) (Canela, A., et al., Proc. Natl. Acad. Sci. USA, 2007, 104(13):5300-5).
U.S. Pat. No. 7,695,904 (Cawthon) describes methods for amplifying target nucleic acids using nucleic acid primers designed to limit non-target nucleic acid dependent priming events. The methods permit amplifying and quantitating the number of repetitive units in a repetitive region, such as the number of telomere repetitive units. The patent also refers to determining average telomere length of an organism by qPCR method.
Thus, despite advances in materials and methods pertaining to telomeres, there remains a need for improved methods and materials for determining measures of short telomere abundance in a population of chromosomes and the use of these measures to determine measures of health and effects of interventions that increase or decrease telomere length and, hence, increase or decrease health, or conversely decrease or increase risk of future disease or death, respectively. These needs and other needs are addressed by the present invention.